The present invention is directed to a novel strain of feline immunodeficiency virus (FIV) and to a variety of mutated forms of this virus. Compositions and methods are disclosed that can be used in the protection of animals from lentiviral associated disease.
Feline immunodeficiency virus (FIV) infection in cats results in a disease syndrome that is similar to that caused by human immunodeficiency virus-1 (HIV-1) infection in humans. Disease progression begins with a transient acute phase (8-10 weeks), followed by a prolonged asymptomatic phase (lasting from weeks to years) and a terminal symptomatic phase (Ishida et al., 1990, Jpn. J. Vet. Sci. 52:645-648). Viral load in plasma has been demonstrated to correlate with disease stage in infected cats and can be used to predict disease progression in accelerated FIV infection (Diehl et a., 1996, J. Virol. 70:2503-2507).
Structurally, the FIV provirus contains two long terminal repeats (LTRs), one at either end of the genome (Talbott et al., 1989, Proc. Nat""l Acad. Sci. USA 86:5743-5747). There are three large open reading frames (Gag (group antigens); Pol (polymerase); and ENV (envelope)) and three small open reading frames encoding regulatory proteins (Rev (regulator of expression of virion, a protein that binds to xe2x80x9cRRExe2x80x9d elements present in all viral transcripts and promotes their translocation from the nucleus to the cytoplasm of infected host cells); Vif (virion infectivity factor); and ORF(2) (open reading frame 2)). The Gag precursor polypeptide of FIV is processed into three mature structural proteins: a matrix protein (MA), a capsid protein (CA), and a nucleocapsid protein (NC). The Pol gene encodes four enzymatic proteins: a protease (PR), a reverse transcriptase (RT), a deoxyuridine triphosphatase (DU), and an integrase (IN). Finally, the ENV precursor polypeptide is processed into two envelope proteins: a surface protein (SU) and a transmembrane (TM) protein.
There have been several attempts to develop a safe and effective vaccine to FIV. Matteucci found that cats inoculated with a conventional fixed cell vaccine were protected from challenge with homologous virus despite an apparent absence of neutralizing antibodies after vaccination. Protection was found to be short-lived and difficult to boost (Matteucci et al., 1996, J. Virol. 70:617-622; Matteucci et al., 1997, J. Virol. 71:8368-8376). These results may be contrasted with those of Verschoor, who observed no protection after the administration of a fixed cell vaccine (Verschoor et al., 1995, Vet. Immunol. Immunopathol. 46:139-149).
Another type of conventional vaccine that has been tested is comprised of whole, inactivated FIV virus. Yamamoto reported that greater than 90% of cats administered a vaccine of this type exhibited essentially complete protection against homologous challenge and slight protection against heterologous virus (Yamamoto et al., 1993, J. Virol. 67:601-605). Both humoral and cellular immunity against FIV were induced and a high level of anti-ENV, anti-core and virus neutralizing (VN) antibodies were observed in the vaccinated cats. In contrast, vaccination of cats with inactivated whole FIV incorporated into immune stimulating complexes (ISCOMS) failed to protect animals from homologous challenge (Hosie et al., 1992, Vet. Immunol. Immunopathol. 35:191-197).
Another approach to vaccine development has involved the use of subunit vaccines containing recombinant core protein, synthetic V3 peptides, and recombinant ENV protein (Elyar et al., 1997, Vaccine 15:1437-1444). Although significant levels of antibodies were induced by such vaccines, none were identified that could protect cats against homologous FIV challenge (Huisman et al., 1998, Vaccine 16:181-187; Flynn et al., 1997, J. Virol. 71:7586-7592; Tijhaar et al., 1997, Vaccine 15:587-596). The results suggest that it is likely to be difficult to obtain protective immunity against FIV using subunit type vaccines.
Recently, Cuisinier reported on tests conducted on a DNA vaccine for FIV (Cuisinier et al., 1997, Vaccine 15:1085-1094). Cats were vaccinated with a plasmid carrying FIV structural genes, including ENV and p10. Although strong humoral immune responses were observed, all cats eventually succumbed to homologous challenge.
The present invention is based, in part, upon the isolation and characterization of a new strain of feline immunodeficiency virus, designated herein as FIV-141 and deposited as ATCC No. VR-2619. The complete genomic sequence of the virus has been determined and is distinct from all other known FIV sequences. A plasmid encoding FIV-141 has been deposited as ATCC No. 203001.
A. Compositions and Methods Based upon the FIV-141 Virus
In its first aspect, the present invention is directed to a substantially purified FIV-141 virus having a genomic sequence corresponding to that of SEQ ID NO:1, to host cells infected with the virus and to progeny virus produced in the host cells. The term xe2x80x9csubstantially purifiedxe2x80x9d means that FIV-141 has been separated from all other strains of virus and, particularly, from all other strains of FIV. Host cells are typically cells grown in in vitro culture. Host cells that may be used for growing virus include peripheral blood mononuclear cells (PBMCs). Progeny virus may be isolated using standard procedures as discussed below. The present invention further provides a substantially purified virus having a nucleotide sequence which is a degenerate variant of a nucleotide sequence corresponding to SEQ ID NO:1, as based on the degeneracy of the genetic code, host cells infected with such a virus, and progeny virus produced in the host cells, which are useful for all of the purposes disclosed herein for the substantially purified FIV-141 virus having a genomic sequence corresponding to that of SEQ ID NO:1, and for which all of the disclosure provided herein below is equally applicable.
The FIV-141 virus and host cells infected with the virus can be used to infect animals for the purpose of inducing the production of antibodies that react preferentially with one or more strains of FIV. xe2x80x9cPreferential bindingxe2x80x9d of antibodies, as used herein, refers to an antibody having at least a 100-fold greater affinity for FIV than for any other virus or non-FIV protein. Antibodies may be generated in any of the animals commonly used for this purpose (such as, e.g., mice, rabbits, goats, or sheep) but, preferably, antibodies will be made in domestic cats. When virus is used to induce antibody production, it may, if desired, be inactivated prior to infection. Inactivation procedures may involve treating the virus with formalin, paraformaldehyde, phenol, lactopropionate, ultraviolet light, heat, psorlens, platinum complexes, ozone or other viricidal agents. When host cells expressing FIV-141 are used to induce antibody production, the cells may be fixed prior to infection. Typically, this will involve treating the cells with paraformaldehyde as described herein, but other methods may also be employed. Antibodies made to FIV-141 are themselves included within the scope of the invention and may be purified using techniques well known in the art (see, e.g., Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.).
In another aspect, the invention is directed to a whole virus vaccine comprising inactivated FIV-141 virus, or an inactivated virus encoded by a degenerate variant of a nucleic acid molecule having a nucleotide sequence corresponding to SEQ ID NO:1. An immune response may be induced in a cat by administering this vaccine at a dosage and for a duration sufficient to induce protective immunity against subsequent infection with FIV-141. Typically, the vaccine will be administered parenterally with two or more inoculations being given at intervals of, e.g., two to eight weeks. The invention also includes a fixed cell vaccine, which is comprised of a host cell infected with the FIV-141 virus or a degenerate variant thereof. Administration of this vaccine will follow the same general procedures as used for the whole virus vaccine. Standard procedures well known in the art may be used to optimize immunization protocols.
B. Compositions and Methods Based upon FIV-141 Genomic Nucleic Acid
In another aspect, the present invention is directed to a substantially purified nucleic acid molecule (DNA or RNA) having a sequence corresponding to that of SEQ ID NO:1 or a degenerate variant thereof. As used in this context, xe2x80x9csubstantially purifiedxe2x80x9d means that the desired product is essentially free from contaminating cellular components. A xe2x80x9csubstantially purexe2x80x9d nucleic acid will typically comprise at least 85% of a sample, with greater percentages being preferred. Contaminants may include proteins, carbohydrates or lipids. One method for determining the purity of a nucleic acid is by electrophoresing a preparation in a matrix such as polyacrylamide or agarose. Purity is evidenced by the appearance of a single band after staining. Other methods for assessing purity include chromatography and analytical centrifugation. The FIV-141 nucleic acid may be used in place of the whole virus to transfect host cells and to thereby induce the production of progeny virus or viral proteins.
The invention also encompasses methods of inducing the production of antibodies to FIV-141 by injecting nucleic acid directly into an animal or by administering host cells transfected with the nucleic acid. As with the procedures discussed above in connection with the whole virus, host cells may be fixed prior to administration. Antibodies may be substantially purified from animals and used in assays designed to detect the presence of FIV in culture medium or in a biological fluid. A xe2x80x9csubstantially purifiedxe2x80x9d antibody will typically comprise at least 70% of protein in a sample, with greater percentages being preferred.
Host cells transfected with FIV-141 genomic DNA or a degenerate variant thereof may also be used in a vaccine for immunizing cats. If desired, such cells may be fixed to reduce viral infectivity, e.g., by treatment with an agent such as paraformaldehyde. Vaccines made in this manner may be used to induce an immune response in a cat. The vaccine may be administered using a standard immunization protocol optimized for the induction of protective immunity against subsequent infection with FIV-141 or, if desired, some other strain of FIV.
C. Attenuated FIV-141 Virus and Vaccines
Before a whole virus can be administered to an animal as a vaccine, it must be converted into a non-pathogenic form. As discussed above, this may be accomplished by inactivating the virus or fixing host cells. An alternative method involves introducing mutations into the virus to transform it into an attenuated form. The phrase xe2x80x9cattenuated virusxe2x80x9d as used in this context, refers to a virus that has substantially reduced infectivity compared to its wild type counterpart. Infectivity may be measured in PBMCs, as described in the Examples section herein below.
Thus, the invention is directed to an attenuated FIV-141 virus, or degenerate variant thereof, that exhibits significantly reduced infectivity for feline T lymphocytes relative to the wild type (i.e., non-mutated) virus. The attenuated virus is produced by mutating one or more genes in the FIV-141 genome or degenerate variant thereof selected from the group consisting of Vif, MA, ORF(2), ENV, CA, NC, SU, TMf, CT, IN, DU, V3/4, V7/8 and RRE. Appropriate mutations for each of these genes are described herein. Examples of several specific mutations that may be used in making attenuated viruses include MA del, ENV del, V3/4 del, V7/8 del, TMF del, CT del, Vif del, Vifc del, Vifn del, ORF(2) del, CA del, NC del, IN del, DU del, SU del, and RRE del. In a preferred embodiment, the attenuated virus comprises a mutation in the ENV gene. In a further preferred embodiment, the attenuated virus comprises a combination of any two or more of the aforementioned mutations. In specific though non-limiting embodiments, the attenuated virus comprises double mutations in any of the following combinations of genes: (i) MA/TMf; (ii) MA/V3/4; (iii) MA/Vif; or (iv) ENV/IN. In a preferred embodiment, the attenuated virus comprises any of the following double deletions: (i) MA del/TMf del; (ii) MA del/V3/4 del; (iii) MA del/Vif del or (iv) ENV del/IN del. In a further preferred embodiment, the attenuated virus comprises at least two mutations, one of which is in the ENV gene such as, e.g., ENV del, with one or more other mutations in any of the other genes of the virus. In a preferred embodiment, the one or more other mutations in the other genes of the virus are in genes selected from the group consisting of IN, CA, NC, Vif and ORF(2).
The invention also encompasses host cells infected with the attenuated virus and the progeny virus produced by such cells. Once produced, the attenuated virus may be purified from host cells using standard procedures.
Antibody production may be induced by infecting an animal with the attenuated virus or, alternatively, infected host cells may be used. If desired, the virus may be inactivated or the host cells fixed prior to administration to an animal and antibodies may be purified from animals using standard procedures.
In addition, the invention encompasses a vaccine that utilizes the attenuated whole virus discussed above or a host cell infected with one of these viruses. Again, the attenuated viruses may be inactivated and the host cells may be fixed. Such treatments may provide added assurance that vaccines will not themselves cause infection. Vaccines based upon one or more attenuated FIV-141 viruses or degenerate variants thereof may be used to induce protective immunity in a cat. Standard immunization protocols may be followed in administering vaccines so as to optimize the induction of protective immunity against subsequent challenge with FIV-141.
D. Compositions and Methods Based upon Mutated FIV-141 Genomic DNA
In another aspect, the present invention is directed to a substantially purified FIV-141 nucleic acid (DNA or RNA) having a sequence corresponding to SEQ ID NO:1, or degenerate variant thereof, but which has been mutated to encode an attenuated virus. Mutations should be to one or more genes selected from the group consisting of Vif, MA, CA, NC, SU, TMf, ORF(2), CT, ENV, Vifn, Vifc, IN, DU, V3/4, V7/8, and RRE, and should be made in such a manner that, upon introduction into a host cell, a virus is made that has significantly reduced infectivity for feline T lymphocytes (or other susceptible cell types) relative to the wild type virus. Examples of several specific mutations that will produce an appropriate attenuated virus are described herein and include: MA del, ENV del, V3/4 del, V7/8 del, TMf del, CT del, Vif del, Vifc del, Vifn del, ORF(2) del, SU del, CA del, NC del, IN del, DU del, and RRE del. In a preferred embodiment, the attenuated virus comprises a combination of any two or more of the aforementioned mutations. In specific though non-limiting embodiments, the attenuated virus comprises double mutations in any of the following combinations of genes: (i) MA/TMf; (ii) MA/V3/4; (iii) MA/Vif; or (iv) ENV/IN. In a preferred embodiment, the attenuated virus comprises any of the following double deletions: (i) MA del/TMf del; (ii) MA del/V3/4 del; (iii) MA del/Vif del or (iv) ENV del/IN del. In a further preferred embodiment, the attenuated virus comprises at least two mutations, one of which is in the ENV gene such as, e.g., ENV del, with one or more other mutations in any of the other genes of the virus. In a preferred embodiment, the one or more other mutations in the other genes of the virus are in genes selected from the group consisting of IN, CA, NC, Vif and ORF(2).
The invention includes host cells transfected with the mutated nucleic acid and FIV progeny virus produced by the host cells.
Also included within the scope of the invention is a method of inducing the production of antibodies to FIV in an animal by injecting either nucleic acid mutated as described above or a host cell that has been transfected with the nucleic acid. If desired, the host cell may be fixed prior to administration. The antibodies produced may be purified using standard methods and used in assays designed to detect the presence of FIV.
The nucleic acid, preferably DNA, which has been mutated, may be used in a vaccine in which it is present at a concentration sufficient to induce protective immunity upon administration to a cat. Alternatively, a vaccine may include a host cell transfected with such DNA and, if desired, the host cell may be fixed after viral proteins are expressed. The vaccines may be administered to a cat at a dosage and for a duration sufficient to induce an immune response in a cat, and immunization protocols may be optimized for inducing protective immunity against subsequent infection by FIV-141.
E. Methods of Making and Using Attenuated Lentiviruses
The methods disclosed herein in connection with the production of attenuated FIV-141 may be effectively applied to the attenuation of other strains of FIV and to other types of lentivirus. A virus that has significantly reduced infectivity relative to its unmutated, wild type counterpart may be made by mutating one or more genes selected from the group consisting of MA, CA, NC, DU, ENV, SU, TMf, CT, Vif, ORF(2), Vifn, Vifc, IN, V3/4, V7/8, and RRE. In a preferred embodiment, the attenuated lentivirus comprises a mutation in the ENV gene. In a further preferred embodiment, the attenuated virus comprises a combination of any two or more of the aforementioned mutations. In specific though non-limiting embodiments, the attenuated virus comprises double mutations in any of the following combinations of genes. (i) MA/TMf; (ii) MA/V3/4; (iii) MA/Vif; or (iv) ENV/IN. In a preferred embodiment, the attenuated virus comprises any of the following double deletions: (i) MA del/TMf del; (ii) MA del/V3/4 del; (iii) MA del/Vif del or (iv) ENV del/IN del. In a further preferred embodiment, the attenuated virus comprises at least two mutations, one of which is in the ENV gene such as, e.g., ENV del, with one or more other mutations in any of the other genes of the virus. In a preferred embodiment, the one or more other mutations in the other genes of the virus are in genes selected from the group consisting of IN, CA, NC, Vif and ORF(2).
Mutations should be designed to eliminate or substantially reduce the activity of the gene product. This may be accomplished by deleting the entire gene or by deleting a large (e.g., one fourth) fraction of the entire gene. The invention encompasses the attenuated lentiviruses made using the disclosed methods, host cells infected with these viruses, and methods of inducing antibody production by injecting the attenuated virus into a mammal. The antibodies may be purified from infected mammals and used in immunoassays. Alternatively, the purified antibodies, or antibody-containing serum derived from animals infected with attenuated virus, may be used to treat a mammal for lentivirus infection.
As used herein, the phrase xe2x80x9cinduction of protective immunityxe2x80x9d, and the like, is used broadly to include the induction of any immune-based response in response to vaccination, including either an antibody or cell-mediated immune response, or both, that serves to protect the vaccinated mammal against the particular lentivirus. The terms xe2x80x9cprotective immunityxe2x80x9d, xe2x80x9cprotective responsexe2x80x9d, xe2x80x9cprotectxe2x80x9d, and the like, as used herein, refer not only to the absolute prevention of any of the symptoms or conditions in the mammal resulting from infection with the particular lentivirus, but also to any detectable delay in the onset of any such symptoms or conditions, any detectable reduction in the degree or rate of infection by the particular lentivirus, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the particular lentivirus. Vaccine preparations according to the present invention should be administered at a dosage and for a duration sufficient to reduce one or more clinical signs and viral load associated with the infection of the mammal. When the lentivirus is a strain of FIV, the mammal treated will be a cat and the signs associated with the infection will include immunological abnormalities such as an abnormally low level of CD4+ T-lymphocytes or an abnormally elevated number of CD8+ T-lymphocytes. Other clinical signs will typically include alopecia, anemia, chronic rhinitis, conjunctivitis, diarrhea, emaciation, enteritis, gingivitis, hematochezia, neurological abnormalities and dermatitis.
The attenuated lentivirus (e.g., an attenuated strain of FIV), or host cells infected with such a virus, may be used in vaccine at a concentration sufficient to induce immunity when administered to a mammal (e.g., a cat). An immune response may then be induced by administering such a vaccine at a dosage and for a duration sufficient to induce protective immunity against subsequent infection by at least one strain of lentivirus.
F. Methods of Making and Using Mutated Lentivirus Nucleic Acid
The present invention is also directed to a method of producing a nucleic acid suitable for use in a vaccine against lentivirus, e.g. FIV, infection. This is accomplished by reverse transcribing the genomic RNA of the lentivirus; cloning the reverse transcript; mutating one or more genes selected from the group consisting of MA, CA, NC, SU, TMf, ORF(2), CT, ENV, Vif, Vifn, Vifc, V3/4, V7/8, IN, DU, and RRE; and then cloning the mutated nucleic acid. Preferably, mutations should be such that, upon introduction into a host cell, an attenuated virus is made that has significantly reduced infectivity relative to lentivirus produced by the unmutated, wild type nucleic acid. In the case of FIV, infectivity should be reduced or eliminated for feline T-lymphocytes.
Mutated lentivirus nucleic acid may be purified and used to transfect host cells, make progeny virus, and make antibody in the same way as described above for FIV-141. In addition, the nucleic acid, or host cells transfected with the nucleic acid, may be incorporated into a vaccine and used to induce protective immunity in a mammal. Preferably, the nucleic acid will encode an attenuated strain of FIV that has significantly reduced infectivity in feline PBMCs, including T-lymphocytes such as FeP2 cells. Under these circumstances, the immune response will be induced in a cat.